Continuing efforts have been made to increase human neonatal survival rates with great progress towards improving the chances of survival for full-term and premature babies. Nutrition plays a key role in determining how well a newborn will thrive and develop. Nutritional information is, of course, provided with artificial formulas for infants. However, human breast milk is generally regarded as superior to formulas, giving infants a significant advantage with regards to growth and development, yet human breast milk is rarely analyzed with regards to fat content, which is directly related to energy content. Such information is critical to the care of premature infants and babies that fail to gain weight at a normal rate.
Many methods, as well as instruments employing such methods, have been developed for determining the fat content of milk. While some of the methods have enjoyed popularity in the dairy industry, none has gained widespread use or acceptance in clinical or health care settings. The Babcock method developed in the late 1800's became a standard procedure for determining milkfat content in the dairy industry. The procedure is somewhat hazardous, involving mixing a raw milk sample with concentrated sulfuric acid followed by heating and centrifugation of the mixture. Further, the Babcock method is not suitable for very small test sample volumes, for example, less than 100 microliters.
U.S. Pat. No. 3,161,768 to Goulden teaches a method of using infrared absorption, measured at the wavelength of absorption at the ester linkages (approximately 5.72 microns), for determining fat content of the disperse phase in an emulsion or suspension, such as milk. Such measurements are significantly affected by differences in diet and even genetic differences in cattle, but more reliable measurements are obtained when infrared absorption is measured at the carbon-hydrogen stretching wavelength (approximately 3.48 microns) as taught in U.S. Pat. No. 4,247,773 to Nexo et al. A disadvantage is that milk must be homogenized to reduce fat particle size in order to obtain meaningful data by such absorptiometric methods.
The International Dairy Federation published a standard procedure (IDF Standard 9C, 1987) for determining fat content of dried milk known as the Rose Gottlieb method. This gravimetric method is complicated, lengthy, and involves the use of solvents. Moreover, the milk sample must first be dried and the method is not suitable for small samples of milk less than 100 ml.
Other methods described for determining fat content include colorimetric methods, based upon a color reaction between milk fat and hydroxamic acid, and analysis of fat content by nuclear magnetic resonance (NMR). These procedures are complicated and require relatively expensive, specialized equipment.
In an attempt to develop a procedure with clinical applications, Lucas et al. described a simplified method for determining the fat and energy content of human milk based upon centrifugation of a small sample collected in a standard hematocrit capillary tube for fifteen minutes. See Lucas et al., Br. Med. J. 1:1018, 1978. The length of the cream layer is measured and calculated as a percentage of the total length of the milk column, for example, using a standard hematocrit-measuring card to determine the volume percentage of fat, referred to as a creamatocrit. Use of a hematocrit reader card requires the user to visually align several interfaces at once and then use the determined fat content to calculate estimated caloric content. Alternatively, calipers can be used to measure the cream column and the total length of the centrifuged milk specimen in the capillary tube, and the measurements obtained can be used to calculate the percentage of milkfat, with further calculations needed to determine estimated caloric content. In yet another method, micro-capillary readers can be used to mechanically determine fat content by manually aligning the capillary tube after centrifugation with an index mark, then aligning two rotating disks with the total length and the cream column; when a reading is determined, the user then must subtract the number from one hundred to obtain the percentage of milkfat and then perform further calculations to estimate caloric content.
Although the creamtocrit method represents a significant simplification in relation to other methods for determining milkfat content, it, too, has failed to secure broad acceptance in clinical settings. Further improvements directed towards shortening centrifugation time and to simplifying measurements and calculations of fat and energy content are needed in order for the creamatocrit technique to enjoy widespread use, particularly in clinical and public health care settings where the determination of fat and energy content of human milk is critical to neonatal and infant nutrition, as well as veterinary and research applications for non-human mammals.